Lack of association between putative transporter gene polymorphisms in Plasmodium falciparum and chloroquine resistance in imported malaria isolates from Africa

Abstract

Background: Plasmodium falciparum drug resistance represents a major health problem in malaria endemic countries. The mechanisms of resistance are not fully elucidated. Recently, an association between putative transporter gene polymorphisms and in vitro response to chloroquine (CQ) and quinine has been reported in culture-adapted, cloned isolates from various geographical origins. However, this was not confirmed in another study performed on isolates from a defined region in Thailand.

Methods: This study tried to find an association between putative transporters gene polymorphisms with in vitro response to CQ and pfcrt genotype in isolates originating from various African countries. To avoid biases of parasites adaptation in culture, fresh isolates obtained from symptomatic, malaria-infected travellers returning from Africa to France were used. Monoclonal isolates included in the study were selected using a msp-2 fragment analysis method. In vitro susceptibility to CQ, single nucleotide polymorphisms and microsatellite polymorphisms in pfcrt, pfmdr1 and six putative transporter genes were established in 27 isolates and three reference strains.

Results: Polymorphism of pfcrt at positions 76 and 220 showed a significant association with in vitro chloroquine resistance (P < .02 and P < .05 respectively). Polymorphism of pfmdr1 at position 86 showed an equally significant association with in vitro chloroquine response (P < .05). No association was found between SNPs or microsatellite polymorphisms of putative transporter genes and in vitro CQR or pfcrt genotype in imported malaria isolates from Africa.

Conclusion: The previously described association between putative transporter gene polymorphisms and in vitro response to chloroquine (CQ) was not confirmed in the present study.

Figure 1

Microsatellite allelic frequencies of pfcrt and putative transporter genes in 27 P. falciparum isolates according to pfcrt 76 allele and chloroquine response phenotype. Microsatellite allelic relative frequencies are given according to pfcrt K76 (wild allele, orange bars) or pfcrt K76T (mutant allele, blue bars) (Figure a, c, e, g and i), or according to inhibitory concentration values (IC₅₀) for chloroquine (susceptible isolates, yellow bars; resistant isolates, green bars) (Figure b, d, f, h and j). The relative frequency of the various allelic forms within each haplotype subgroup is indicated. The microsatellites have either (AAAT)n alleles or (TA)n alleles and they are classified according to the number (n) of repeats. Figure a, b: pfcrt microsatellite in intron 4; Figure c, d: G7 microsatellite; Figure e, f: G25 microsatellite in intron 3; Figure g, h: G25 microsatellite in intron 4; and Figure i, j: G55 microsatellite. Motif 1: TAATATTATAATAT(TA)9T Motif 2: TAATAT(TA)11TMotif 3: TAATAT(TA)10T Motif 4: TAATAT(TA)9T.